Characterisation of the porcine cytokines which activate the CD131ßc common sub-unit, for potential immune-augmentation.

Early acting cytokines and growth factors such as those of the CD131 ßc subunit, may offer an alternative method to the current use of antibiotics and chemicals such as anthelmintics in maintaining Porcine (Po) health. Thus far, the recombinant Po (rPo) Granulocyte-macrophage colony-stimulating factor (GM-CSF), rPo interleukin-3 (IL-3) and rPo interleukin-5 (IL-5) proteins have been identified and cloned and the biological activity of each cytokine has been confirmed in vitro, however, in vivo immune system regulation and hematopoietic stem cell (HSC) augmentation are regulated by numerous cytokines and cellular signals within the bone marrow (BM) niche. In order to quantify the use of recombinant cytokines in augmenting the immune response, it is necessary to determine the stages of hematopoiesis induced by each cytokine and possible areas of synergy requiring further investigation. Here we used the chemotherapeutic agent 5-fluorouracil (5-FU), to chemically induce a state of myelosuppression in young pigs. This allowed for the monitoring of both the autologous BM reconstitution and recombinant cytokine induced BM repopulation, precursor cell proliferation and cellular differentiation. The recombinant cytokines PoGM-CSF, PoIL-3 and PoIL-5 were administered by intramuscular injections (i.m.) following confirmation of 5-FU induced leukocytopenia. Blood and BM samples were collected and then analysed for cell composition. Statistically significant results were observed in several blood cell populations including eosinophils for animals treated with rPoIL-5, rPoGM-CSF and basophils for animals treated with rPoIL-3. BM analysis of CD90+ and CD172a+ cells confirmed myelosuppression in week one with significant results observed between rPoIL-3 and the 5-FU control group in week two and for the rPoGM-CSF group in week three. These results have demonstrated the effects of each of these rPo cytokines within the hematopoietic processes of the pig and may demonstrate similar outcomes in other mammalian models including human.

Avian cytokines - the natural approach to therapeutics.

While the effective use of antibiotics for the control of human disease has saved countless lives and has increased life expectancy over the past few decades, there are concerns arising from their usage in livestock. The use of antibiotic feed additives in food production animals has been linked to the emergence in the food chain of multiple drug-resistant bacteria that appear impervious to even the most powerful antimicrobial agents. Furthermore, the use of chemical antimicrobials has led to concerns involving environmental contamination and unwanted residues in food products. The imminent banning of antibiotic usage in livestock feed has intensified the search for environmentally-friendly alternative methods to control disease. Cytokines, as natural mediators and regulators of the immune response, offer exciting new alternatives to conventional chemical-based therapeutics. The utilisation of cytokines is becoming more feasible, particularly in poultry, with the recent cloning of a number of avian cytokine genes. Chickens offer an attractive small animal model system with which to study the effectiveness of cytokine therapy in the control of disease in intensive livestock. In this report we will review the status of avian cytokines and focus on our recent studies involving the therapeutic potential of chicken interferon gamma (ChIFN-gamma) as a vaccine adjuvant and a growth promoter.

Coadministration of IFN-gamma enhances antibody responses in chickens.

The development of new generation vaccines has focused on the use of natural immunologic adjuvants that are capable of enhancing a protective immune response. The use of cytokines as immunomodulators in livestock animals, particularly poultry, is becoming more feasible with the recent cloning of several cytokine genes and the progression of new delivery technologies, such as live vectors and DNA delivery. Given that chickens are reared under intensive conditions that are conducive to infection by opportunistic pathogens, the primary mechanism for disease control in poultry is early and effective vaccination. However, many poultry vaccines offer only short-term protection or give nonuniform responses within flocks. We have developed a model system with which to measure the adjuvant potential of cytokines in chickens. This involves measuring antibody levels following coadministration of chicken interferon-gamma (Ch-IFN-gamma) with sheep red blood cells (SRBC). Groups of SPF and commercial broiler birds were injected with two different doses of SRBC with and without coadministration of Ch-IFN-y. Three weeks later, all birds were boosted with SRBC alone. Sera were collected weekly and anti-SRBC antibody titers (total Ig and IgG) were determined by hemagglutination. Priming Ch-IFN-gamma resulted in enhanced primary and secondary (IgG) antibody responses that persisted at higher levels when compared with birds that received SRBC alone. Second, coadministration of Ch-IFN-y allowed a 10-fold lower dose of antigen to be as effective as a high dose of antigen that was given without Ch-IFN-gamma. Third, treatment with Ch-IFN-y resulted in an increase in the proportion of birds responding to antigen challenge. These results suggest the potential use for Ch-IFN-gamma as a vaccine adjuvant.

In vivo effects of chicken myelomonocytic growth factor: delivery via a viral vector.

We have constructed a recombinant fowlpox virus (FPV) that expresses chicken myelomonocytic growth factor (cMGF). Administration of this construct (fp/cMGF) to 1-day-old chicks resulted in a marked and sustained increase in the number of circulating blood monocytes compared with chicks infected with the parental FPV strain (fp/M3). Blood monocyte numbers were elevated within 4 days of fp/cMGF infection, reached maximal levels at day 9, and returned to normal levels by day 16. During the peak response, approximately 35% of blood leukocytes were monocytes, compared with 4 to 7% in uninfected control birds. Infection with fp/M3 also resulted in a detectable increase in monocyte numbers; however, the effect was less dramatic. Compared with fp/M3, fp/cMGF consistently induced two- to threefold higher monocyte numbers, and the period of monocytosis was longer (10 vs 5 days). No other specific changes in white blood cell populations were observed. Associated with the increase in the number of monocytes was an increase in their state of activation, as measured by the ability to produce nitric oxide (NO) and to phagocytose latex beads. Blood monocytes from birds infected with fp/cMGF produced about 6 times as much NO per cell compared with monocytes from fp/M3-infected birds. Monocytes from normal birds failed to produce detectable levels of NO. Furthermore, cMGF treatment specifically induced enhanced phagocytic activity in blood monocytes. Overall, these results indicate that viral vectors are suitable for the delivery of biologically active cytokines and that they allow an assessment of cytokine activities in vivo.

Isolation of high frequency of recombination donors from Tn5 chromosomal mutants of Pseudomonas putida PPN and recalibration of the genetic map.

A Tn5 loaded derivative of the IncP-10 plasmid R91-5 (pMO75) was used as a suicide vector to generate random chromosomal insertion mutations in Pseudomonas putida PPN. Reintroduction of pMO75 into such mutants resulted in integration of the plasmid at the site of Tn5 insertion, giving rise to two classes of high frequency of donors recombination (Hfr) donors, transferring chromosome at high frequency (greater than 10(-1) per donor cell) in opposite directions. Consequently, Tn5 induced auxotrophic mutations could be equated with or distinguished from previously mapped mutations, and closely linked markers ordered, on the basis of marker recovery using the two classes of Hfr donor. The isolation of many new transfer origins allowed more accurate time-of-entry analysis than previously possible and resulted in the reduction of the genetic map from 103 min to 88 min.